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What Can We Learn about Cholesterol’s Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential Stanislav G. Falkovich, Hector Martinez-Seara, Alexey M. Nesterenko, Ilpo Vattulainen, and Andrey A. Gurtovenko J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b02123 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016

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The Journal of Physical Chemistry Letters

What Can We Learn about Cholesterol’s Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential Stanislav G. Falkovich,† Hector Martinez-Seara,‡,¶ Alexey M. Nesterenko,§ Ilpo Vattulainen,¶,k,⊥ and Andrey A. Gurtovenko∗,†,# Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoi Prospect V.O. 31, St.Petersburg, 199004 Russia, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám¸estí 542/2, 166 10 Praha 6, Czech Republic, Department of Physics, Tampere University of Technology, P.O. Box 692, FI-33101 Tampere, Finland, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, 1/40, 119991 Moscow, Russia, Department of Physics, P. O. Box 64, FI-00014 University of Helsinki, Finland, MEMPHYS – Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark, and Faculty of Physics, St.Petersburg State University, Ulyanovskaya str. 3, Petrodvorets, St.Petersburg, 198504 Russia E-mail: [email protected];Web:biosimu.org

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Abstract Cholesterol is abundant in the plasma membranes of animal cells and is known to regulate a variety of membrane properties. Despite decades of research, the transmembrane distribution of cholesterol is still a matter of debate. Here we consider this outstanding issue through atomistic simulations of asymmetric lipid membranes, whose composition is largely consistent with eukaryotic plasma membranes. We show that the membrane dipole potential changes in a cholesterol dependent manner. Remarkably, moving cholesterol from the extracellular to the cytosolic leaflet increases the dipole potential on the cytosolic side, and vice versa. Biologically this implies that by altering the dipole potential, cholesterol can provide a driving force for cholesterol molecules to favor the cytosolic leaflet, in order to compensate for the intramembrane field that arises from the resting potential.

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dipole potential intramembrane field .

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To whom correspondence should be addressed Institute of Macromolecular Compounds RAS ‡ Institute of Organic Chemistry and Biochemistry ¶ Tampere University of Technology § Moscow State University k University of Helsinki ⊥ University of Southern Denmark # St. Petersburg State University †

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Cholesterol molecules are vital for regulating a variety of cell membrane properties such as in-plane structural organization, fluidity, and lateral dynamics. 1,2 Given the outstanding importance of cholesterol, its effects on cell membrane structures have been explored for decades. The transmembrane distribution of cholesterol has, however, remained an open issue. One is tempted to assume that in eukaryotic plasma membranes cholesterol would reside mainly in the extracellular membrane leaflet, 3 since cholesterol is known to interact very favorably with sphingolipids, 4 which in turn are known to be most abundant on the extracellular side. However, there are also data that suggest cholesterol to reside mainly in the cytosolic leaflet. 5–7 Since these studies have been carried out under quite complicated conditions due to the use of, e.g., detergents and cold temperatures, the idea that cholesterol would reside mainly on the cytosolic side has not received general approval. Resolving this issue would be a quite simple feat, if cholesterol’s transmembrane distribution could be studied enzymatically, such as with phospholipids (via phospholipases) or sphingolipids (using sphingomyelinases). However, due to the lack of appropriate enzymes this has not been possible. The distribution of cholesterol across plasma membranes has therefore remained an outstanding open question. 8 Here we approach this problem from a different perspective. We ask ourselves how changes in cholesterol’s transmembrane distribution influence the membrane’s dipole potential. This potential develops within the membrane-water interface region and is considered to be essential for conformations and the function of membrane proteins, and more generally for interactions between a lipid membrane and other biological molecules embedded in a membrane. 9–11 It can also be relevant for lipid-mediated hydrogel-colloid interactions. 12 Experimental studies have demonstrated that cholesterol leads to an increase of the dipole potential in model phosphatidylcholine membranes, the effect being more pronounced for saturated lipids as compared to unsaturated counterparts. 13–16 Cholesterol has been shown to induce a similar effect also in cell membranes and in reconstituted membranes composed of natural membrane lipids. 13,14

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However, the effect of cholesterol on the dipole potential of the separate extracellular and cytosolic leaflets of cell membranes is mostly unknown. It is established that the leaflets of plasma membranes differ in their lipid composition. 17 In general, in animal cells phosphatidylcholine (PC) and sphingomyelin (SM) lipids are localized mostly in the extracellular leaflet, whereas phosphatidylethanolamine (PE) and (anionic) phosphatidylserine (PS) are predominant lipids in the cytosolic leaflet. 18 Therefore, one can expect that the effect of cholesterol (Chol) could be different in these distinctly different leaflets. Such differences cannot be explored in single-component symmetric model membranes (vesicles) that are often employed in experimental measurements of the dipole potential. 14–16 Asymmetric lipid membranes would be more appropriate to consider this task, but they are still challenging from experimental point of view. 8 In this work, we use atomistic molecular dynamics (MD) simulations to clarify how changes in cholesterol’s transmembrane distribution influence the dipole potential in asymmetric lipid membranes, whose composition is chosen to match eukaryotic plasma membranes to an adequate degree. Because the exact distribution of cholesterol molecules in cell membranes is not known, we vary the cholesterol distribution systematically. We also account for cholesterol flip-flops that are known to be frequent. 19,20 Therefore, instead of studying an asymmetric membrane directly, we consider dipole potential profiles in membranes comprised of a pair of leaflets taken from the opposite leaflets of corresponding asymmetric membranes. This approach is justified given that the monolayer’s dipole potential develops mostly within the lipid-water interface and is not affected to a large degree by the hydrophobic acyl chain region. 21 Two types of lipid bilayer mixtures were studied, see Figure 1. The bilayers comprised of palmitoyl-oleoyl-phosphatidylcholine (POPC) and SM were considered as a model of the extracellular leaflet. The cytosolic leaflet of plasma membranes is modelled by a bilayer composed of palmitoyl-oleoyl-phosphatidylethanolamine (POPE) and palmitoyl-oleoylphosphatidylserine (POPS). POPC, POPE, and SM are zwitterionic (neutral) lipids with

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polar lipid head groups, while POPS is anionic and characterized by a net charge (-1e). PCSM bilayers consisted of 64 PC and 64 SM lipids, together with 0, 32, or 64 Chol molecules, leading to three PC-SM-Chol systems with respective molecular ratios 64:64:0, 64:64:32, and 64:64:64 (corresponding Chol molar concentrations were 0, 20, and 33 mol %, respectively). The numbers of cholesterol molecules were the same in PE-PS bilayers but the numbers of PE and PS lipids were slightly adjusted to ensure that the leaflet areas of PC-SM and PE-PS bilayers would match. This was necessary to eliminate possible finite size effects in asymmetric membranes assembled from monolayers with different lipid compositions (see refs. 22 and 23). Consequently, we considered PE-PS-Chol bilayer systems with respective molecular ratios 118:30:0, 100:26:32, and 88:22:64 (or with Chol molar concentrations 0, 20, and 37 mol %). The number of water molecules ranged between 5100 and 7700, thus the hydration of the lipids was high. Simulations of every bilayer system were repeated twice: (i) under salt-free conditions and (ii) in a saline solution at a physiological concentration (∼ 150 mM) and an appropriate electrolyte composition 24–26 (NaCl for PC/SM/Chol bilayers and KCl for PE/PS/Chol bilayers). The results are based on two force fields whose outcomes were compared on equal footing for validation. All phospholipids and cholesterol molecules were first described by the unitedatom force-field by Berger et al. 27 Water was then represented by the simple point charge (SPC) model 28 and all systems were simulated in the NpT ensemble at the physiological temperature (T = 310 K) and pressure (1 bar). The velocity-rescaling thermostat 29 was used to control the temperature, while the Parrinello-Rahman scheme 30 was employed to keep the pressure constant through a semi-isotropic coupling. The time step used was 2 fs. All 12 bilayer systems were simulated for 1 µs each; the last 300 ns were used for analysis. Moving on, for validation and to explore the sensitivity of the simulation results to the force field employed, the simulations of all systems were repeated with the all-atom lipid force field CHARMM36. 31 The lipid composition and simulation conditions were kept as close as possible to those used in Berger simulations. The GROMACS suite was used for

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both simulation sets (version 4.5.6 for Berger lipids 32,33 and version 5.0.4 for CHARMM36 lipids). 34 Below we discuss the results based on the Berger force field unless mentioned otherwise. The electrostatic potential of the lipid membranes was calculated from a MD simulation trajectory by integrating the 1D Poisson equation twice, as outlined in ref. 35. Each potential profile was then symmetrized with respect to the center of mass (COM) of the membrane, resulting in the electrostatic potential profile of one leaflet. Figure 2 depicts how these potential profiles for PC-SM-Chol and PE-PS-Chol monolayers are combined to mimic the electrostatic potential of an effectively asymmetric membrane. The electrostatic potential difference between the membrane’s COM and the bulk water phase (taken at a distance of 4 nm from the COM of a membrane) defines the potential known as the boundary potential (ψb ). This boundary potential consists of the dipole potential (ψd ) and the surface potential (ψs ) that always appears at charged surfaces. 36,37 The surface potential was evaluated from the potential of mean force of ions at the closest plane corresponding to a zero surface excess of water following a procedure outlined in ref. 38. Finally, the dipole potential was calculated as ψd = ψb − ψs . Figure 2 (top) and Table 1 highlight that Chol makes a difference for the membrane dipole potential: cholesterol molecules increase the boundary potential of a membrane to a significant extent. As the effect of cholesterol on the surface potential is minor (≤ 10 mV, see Table 1), this result also holds for the dipole potential. We find that adding 20 and 33 mol% of cholesterol to PC-SM membranes increases the dipole potential by 61 and 48 mV, respectively. The same trend is found for PE-PS membranes, where adding 20 and 37 mol% of cholesterol to a membrane increases the dipole potential by 55 and 118 mV, respectively. Cholesterol clearly increases the membrane’s dipole potential in a manner that is dependent on membrane lipid composition. Under physiological conditions membranes are exposed to salt, whose composition is different on the extracellular and cytosolic sides. Figure 2 (bottom) and Table 1 demonstrate

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the influence of adding 150 mM of NaCl (KCl) to PC-SM-Chol (PE-PS-Chol) membrane systems with varying content of cholesterol. The overall effect of cholesterol remains unchanged: cholesterol gives rise to a substantial increase in the membrane dipole potential, although the increase is here smaller as compared to salt-free systems. This is an important conclusion given that salt-free conditions do not occur under relevant experimental circumstances (e.g. in experiments done in vivo). In PC-SM-Chol membranes the increase due to 20 and 33 mol% of cholesterol is 39 and 23 mV, respectively. The corresponding changes for PE-PS-Chol membranes with 20 and 37 mol% of cholesterol are 26 and 76 mV, in respective order. While the effect of cholesterol is strong, the influence of salt is less pronounced. Typically the absolute values of the dipole potential are not affected much by salt ions. 39 We can draw largely the same conclusion given that Table 1 indicates the dipole potential to change less than ∼ 20 – 25 mV due to added salt. The only exception is the cholesterol-free PC-SM membrane in which adsorption of sodium ions gives rise to membrane compaction. 26 The surface potential, on the other hand, is quite sensitive to both the type of salt cations and the ionic strength. Cations of different types have different affinities to anionic phospholipids, 37 while the ionic strength affects the surface potential through a change of the Debye screening length. Both effects are well known and often analyzed in terms of the Gouy-Chapman-Stern theory, see e.g. ref 36 for a review. While a detailed description of the effects of salt ions on the membrane’s electrostatic potential is beyond the scope of this paper, let us mention that the observed drop in the surface potential of anionic PE-PS-Chol bilayers (Tables 1 and S1) is a signature of a salt-induced decrease in the electrostatic screening length in the bilayer system. For validation, we repeated the above discussed simulations and the analysis using the CHARMM36 force field. 31 Figures S1 and S2 (see the Supporting Information) show that the conclusions made above are not sensitive to the choice of the force field. In all considered systems, the additional simulations also revealed that cholesterol elevates the membrane

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dipole potential (Table S1). The cholesterol dependent effect with CHARMM36 was stronger compared to simulations with the Berger force field, which stems from a different treatment of lipid hydrocarbon chains in the two force fields. 27,31 How cholesterol gives rise to the observed changes in membrane dipole potential? To answer this question, we calculated component-wise contributions to the boundary potential profiles depicted in Figure 2. In salt-free systems the contribution of zwitterionic lipids to the potential decreases progressively with increasing cholesterol content, while the contribution of cholesterol (arising from its hydroxyl group) increases, see Tables S2 and S4. Perhaps surprisingly, the drop in the contributions of zwitterionic lipids is not related to the re-orientation of their head groups; the tilt angle between the lipids’ PN vector and the outward bilayer normal is not sensitive to cholesterol concentration (Tables S3 and S5). Instead, it is likely caused by the “dilution” of polar head groups on the bilayer surface: cholesterol molecules increase the distance between zwitterionic lipids and reduce their surface number density, see Tables S3 and S5. Correspondingly, the contribution of water molecules (and anionic lipids together with counterions in the case of PE-PS-Chol systems) that compensates the contribution of zwitterionic lipids also decreases. Meanwhile, the surface number density of cholesterol increases with its concentration, thereby strengthening cholesterol’s contribution to the boundary potential, see Tables S3 and S5. This molecular-scale picture captures the essence of the cholesterol dependent potential, however additional subtle interactions such as hydrogen bonding between zwitterionic lipids and cholesterol are also expected to play a role and complement the above effect. For instance, in PC-SM-Chol systems the increase in the boundary and dipole potentials is not a linear function of cholesterol content (see Table 1). Overall, the total cholesterol-induced increase in the boundary potential is of the same order of magnitude as the contribution of cholesterol molecules into the potential. This key conclusion remains unchanged upon adding monovalent salt into the bilayer system, see Tables S6 - S9. Although experimental data for the dipole potential of PC-SM-Chol and PE-PS-Chol

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membranes have not been reported yet, we can compare the computational results with experimental data available for phospholipid membranes. The restriction that we have to take into account, though, is the condition that in experiments one cannot measure absolute values of the dipole potential because it is a variant of the Galvani potential. Therefore, the comparison has to be based on cholesterol-induced relative changes in the dipole potential. Then, it was experimentally shown 14 that removing 80 % of cholesterol from membrane vesicles composed of natural membrane lipids (phospholipids, cholesterol, and other lipids extracted from the kidney and brain of eight vertebrate species) resulted in a reduction in the dipole potential of ∼ 50 mV. A similar difference was observed for unsaturated dioleoyl-phosphatidylcholine vesicles with 30 mol% cholesterol. 14 Interestingly, the influence of cholesterol on the dipole potential was found to be much more pronounced for vesicles composed of saturated dimyristoyl-phosphatidylcholine with about 40 mol% cholesterol (more than 400 mV). 14 Despite the differences in lipid compositions (the lipid membrane mixtures considered in this work do not contain saturated lipids), one can conclude that the simulation models correctly reproduce the right ballpark and the trends of the experimentally observed cholesterol-induced changes in the dipole potential, and even quantitative agreement is reasonable. Careful consideration of Figure 2 brings out highly relevant insight regarding the implications of an asymmetric transmembrane distribution and the role of cholesterol in this context. First, it is obvious that the electrostatic potential profiles of asymmetric membranes are also asymmetric, implying that a non-zero difference in the electrostatic potential develops across the membrane in line with previous experimental 40 and computational studies. 22 Second, moving cholesterol from the extracellular leaflet to the cytosolic side increases the boundary potential and, consequently, also the dipole potential on the cytosolic side of a membrane (and vice versa). To emphasize the importance of the non-zero transmembrane potential difference in cell membranes, Figure 3 sketches two relevant scenarios. The boundary potential difference

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develops within the lipid-water interface region, while the potential difference between the water phases on the opposite sides of a membrane is governed by the resting potential. We recall that the resting potential is due to a charge difference in the cytosolic and extracellular compartments and has passive and active components. The former is a Gibbs-Donnantype potential, the latter is generated by the action of Na+ -K+ pumps. 41 When the resting potential is zero (see the left-hand side column in Figure 3), the boundary potential of the extracellular (PC-SM-Chol) leaflet can be either larger or smaller than the boundary potential of the cytosolic (PE-PS-Chol) leaflet. However, in animal cells the resting potential of membranes is not zero (instead it is ∼ 50 – 100 mV), and it is known to be negative on the cytosolic side of a membrane. 42,43 Taking this into account, let us now apply a nonzero resting potential to a membrane, see the right-hand side column in Figure 3. One can conclude that the field inside a membrane is minimized when the boundary potential of the cytosolic (PE-PS-Chol) leaflet exceeds the corresponding boundary potential on the extracellular (PC-SM-Chol) side. As seen from Figure 2, this is indeed the case for most asymmetric membranes where cholesterol molecules are in the cytosolic (PE-PS-Chol) leaflet only. This conclusion also applies to the dipole potential since cholesterol has almost no effect on the surface potential, as discussed above. Due to rapid (passive) translocation of cholesterol between the opposite leaflets, it is often considered that cholesterol might be distributed evenly between the two monolayers, with some preference to the cytosolic leaflet. 7 Theoretical considerations have suggested that cholesterol would be exposed to a driving force towards the cytosolic leaflet to reduce the membrane’s bending energy, due to the presence of PE in the cytosolic leaflet. 8,44 In this work, we have shown that there is yet another potential physical mechanism: given that cholesterol increases the dipole potential, it can provide a driving force for cholesterol molecules to favor the cytosolic leaflet, in order to compensate for the intramembrane field that arises from the resting potential. In native cell membranes it is not only passive cholesterol translocation but also active

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transport that contributes to the transmembrane distribution of cholesterol. The challenge to unravel the transmembrane distribution of cholesterol in native (or native-like) cell membranes therefore remains still to be clarified. Nonetheless, the present results suggest a new paradigm for assessing how cholesterol is distributed in cells. If membrane potential measurements could be performed rapidly enough, one could measure how the membrane potential responds to changes in cholesterol transmembrane distribution. Such experiments would likely be based on detergents to deplete cholesterol in an asymmetric fashion, but despite this subtle issue, experiments could then generate a more solid understanding as to the distribution of cholesterol.

Supporting Information Results of additional MD simulations with an alternative lipid force field (CHARMM36); component-wise contributions to the boundary potential, surface number densities of lipids and cholesterol, and orientation of lipid head groups (Berger lipids).

Acknowledgments. The authors acknowledge resources granted to use the Lomonosov supercomputer at the Moscow State University and the computer cluster of the Institute of Macromolecular Compounds RAS. This work was partly supported by the EU FP7 Staff Exchange Program (FP7-PEOPLE-2011-IRSES) in the framework of the Project 295302 (SPIDER) and by the Russian Foundation of Basic Research through the Grant 16-04-00556a (A.M.N.). I.V. thanks funding granted by the European Research Council (Advanced Grant CROWDED-PRO-LIPIDS) and the Academy of Finland (Center of Excellence program).

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(31) Klauda, J. B.; Venable, R. M.; Freites, J. A.; O’Connor, J. W.; Tobias, D. J.; MondragonRamirez, C.; Vorobyov, I.; MacKerell, A. D.; Pastor, R. W. Update of the CHARMM All-Atom Additive Force Field for Lipids: Validation on Six Lipid Types. J. Phys. Chem. B 2010, 114, 7830–7843. (32) Lindahl, E.; Hess, B.; van der Spoel, D. GROMACS 3.0: A Package for Molecular Simulation and Trajectory Analysis. J. Mol. Model. 2001, 7, 306–317. (33) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comp. 2008, 4, 435–447. (34) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E. GROMACS: High Performance Molecular Simulations Through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX 2015, 1-2, 19–25. (35) Gurtovenko, A. A.; Vattulainen, I. Calculation of the Electrostatic Potential of Lipid Bilayers from Molecular Dynamics Simulations: Methodological Issues. J. Chem. Phys. 2009, 130, 215107. (36) McLaughlin, S. The Electrostatic Properties of Membranes. Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 113–136. (37) Ermakov, Y. A. The Determination of Binding Site Density and Association Constants for Monovalent Cation Adsorption onto Liposomes Made from Mixtures of Zwitterionic and Charged Lipids. Biochim. Biophys. Acta 1990, 1023, 91–97. (38) Nesterenko, A. M.; Ermakov, Y. A. Molecular-Dynamic Simulation of Phospholipid Bilayers: Ion Distribution at the Surface of Neutral and Charged Bilayer in the Liquid Crystalline State. Biochem.-Mosc. Suppl. A: Membr. Cell Biol. 2012, 6, 320–328. (39) Marukovich, N.; McMurray, M.; Finogenova, O.; Nesterenko, A.; Batischev, O.; Ermakov, Y. Interaction of Polylysines with the Surface of Lipid Membranes: The Electrostatic and Structural Aspects. Advances in Planar Lipid Bilayers and Liposomes 2013, 17, 139–166.

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(40) Latorre, R.; Hall, J. E. Dipole Potential Measurements in Asymmetric Membranes. Nature 1976, 264, 361–363. (41) Sperelakis, N. Gibbs-Donnan Equilibrium Potentials. In Cell Physiology Source Book: A Molecular Approach (3rd ed.); Sperelakis, N., Ed.; Academic Press, 2001; pp 243–247. (42) Lodish, H.; Berk, A.; Zipursky, S. L.; Matsudaira, P.; Baltimore, D.; Darnell, J. E. Molecular Cell Biology; W. H. Freeman and Company: New York, 2000. (43) Voet, D.; Voet, J. G. Biochemistry, 3rd ed.; John Wiley & Sons: New York, 2004. (44) Giang, H.; Schick, M. How Cholesterol Could Be Drawn to the Cytoplasmic Leaf of the Plasma Membrane by Phosphatidylethanolamine. Biophys. J. 2014, 107, 2337–2344.

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Table 1: Electrostatic properties of membrane systems considered. System PC-SM-Chol

PC-SM-Chol

PE-PS-Chol

PE-PS-Chol

Cchol [mol %]1 0 20 33 0 20 33 0 20 37 0 20 37

Salt – – – NaCl NaCl NaCl – – – KCl KCl KCl

ψb [mV]2 682 743 730 747 778 761 632 677 740 661 688 739

1

ψs [mV]3 0 0 0 19 11 10 -35 -45 -45 -21 -20 -19

ψd [mV]4 682 743 730 728 767 751 667 722 785 682 708 758

Cholesterol concentration (mol %). Boundary potential (with zero defined in bulk water). 3 Surface potential (the sign of the potential reflects the sign of the surface charge). 4 Dipole potential. 2

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Figure 1: Snapshots of (A) PC-SM-Chol and (B) PE-PS-Chol lipid membranes. POPC shown in red, SM in cyan, POPE in ice-blue, POPS in green, and Chol in yellow; water molecules and ions are not shown.

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0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 -4

PE-PS-Chol

PC-SM-Chol

Cholesterol 0 mol% -- 37 mol% 20 mol% -- 20 mol% 33 mol% -- 0 mol% -3

-2

-1

0

2

1

3

0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 4

z [nm]

Potential [V]

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Potential [V]

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0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 -4

PE-PS-Chol

PC-SM-Chol

KCl

NaCl

Cholesterol 0 mol% -- 37 mol% 20 mol% -- 20 mol% 33 mol% -- 0 mol% -3

-2

-1

0

1

2

3

0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 -0.8 -0.9 4

z [nm]

Figure 2: Combined electrostatic potential profiles of phospholipid membranes with cholesterol vs. distance z from the membranes’ center of mass (Berger lipids). Shown are profiles for PC-SM-Chol (z < 0) and PE-PS-Chol (z > 0) membranes in (top) salt-free and (bottom) saline conditions. For convenience, the potential was chosen to be zero at the membranes’ center of mass. 19



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PC−SM−Chol

PE−PS−Chol

PC−SM−Chol

PE−PS−Chol

PC−SM−Chol

PE−PS−Chol

PC−SM−Chol

PE−PS−Chol

Figure 3: Schematic representation of the electrostatic potential of an asymmetric lipid membrane (right) with and (left) without an external resting potential. Shown here are the situations where the boundary potential of the extracellular PC-SM-Chol leaflet (top) exceeds or (bottom) is smaller than the potential of the cytosolic PE-PS-Chol leaflet. The surface potential is not shown.

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What Can We Learn about Cholesterol's ... - BioSimu.org

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